Endochondral ossification is remarkably similar in diverse biological settings. The remodeling of calcified cartilage into bone can be found in embryonic sterna, vertebrae, and limbs, juvenile long bone development, fracture healing by callus formation, and ectopic bone formation induced by bone morphogenetic proteins. The same process can also be found in pathologic conditions, such as cartilaginous neoplasms, heterotopic ossification, and degenerating articular cartilage. This commonality suggests that mineralizing chondrocytes are committed to the same innate developmental pathway.
During the process of endochondral ossification, chondrocytes undergo a progression of maturational changes, with marked biochemical and physical changes in both the cells and surrounding matrix. These changes are most evident in the growth plate where they are spatially and temporally ordered (Buckwalter et al., J. Bone and Joint Surg., 68A:243-255 (1986); Gibson et al., Cell Biol., 101:277-284 (1985); and Poole, "Cartilage in Health and Disease", Arthritis and Allied Conditions: A Textbook of Rheumatology, 279-333, (1993)). Resting chondrocytes are flat, irregularly-shaped nondividing cells. As these cells enter the cell cycle, they become arranged in columns and undergo the rapid proliferation necessary for long bone growth. Collagen fibrils in the resting and proliferating region of the growth plate are predominantly type II collagen with associated minor collagens type IX and type XI (Buckwalter Clin. Orthop., 172:207-231 (1983) ("Buckwalter"); Oshima et al., Calcif. Tiss. Int., 45:182-192 (1989) ("Oshima"); Castagnola et al., J. Cell Biol., 102:2310-2317 (1986); Liu et al., Dev. Dynamics, 198:150-157 (1993); and Linsenmyer et al., Development, 111, 191-196 (1991)). The matrix is characterized by an abundance of high molecular weight proteoglycans, which have a structural role in addition to preventing calcification (Buckwalter; Dziewiatkowski et al., Calcif. Tiss. Int., 37:560-567 (1985); Kosher et al., Dev. Biol., 118:112-117 (1986); and Chen et al., Calcif. Tissue Int., 37:395-400 (1985)). In the hypertrophic region of the growth plate, proliferation ceases and a significant increase in cell volume, up to 8-fold, occurs. Hypertrophic chondrocytes form arcades and initiate the synthesis of type X collagen, while collagen types II and IX and proteoglycan content decrease. In the most inferior part of the growth plate, adjacent to the metaphysis, the cartilage mineralizes. Hypertrophic chondrocytes in the calcified tissue may undergo apoptosis (Shapiro et al., J. Bone Min. Res., 10(S1):S238 (1995); Fujita et al., Trans. Ann. Mtg. Othop. Res. Soc., 20:470 (1995); and Farnum et al., Trans. Ann. Mtg. Othop. Res. Soc., 20:77 (1995)), partially convert to an osteoblastic phenotype (Cancedda et al., J. Cell Biol., 117:427-435 (1992)), or remain quiescent until resorption by the invading blood vessels. The signals necessary for calcification are poorly understood, but calcification appears to be effected through the production of matrix vesicles, which contain alkaline phosphatase, phospholipase A.sub.2, NTP-pyrophosphohydrolase, calcium, phosphate, and matrix metalloproteases (Dean et al., Calcif. Tissue Int., 50:342-349 (1992); Lewinson et al., J. Histochem. and Cytochem., 30:261-26 (1982); Wuthier et al., Cal. Tissue Int., 24:163-171 (1977); and Watkins et al., Biochem. Biophys. Acta, 631:289-304 (1980)). The calcified cartilage serves as a scaffold for vascular invasion and deposition of the primary spongiosa.
A variety of cell culture models have been utilized to study the developmental changes associated with endochondral ossification. Embryonic chondrocytes from sterna (Leboy et al., J. Biol. Chem., 264:17281-17286 (1989) ("Leboy"); Sullivan et al., J. Biol. Chem., 269:22500-22506 (1994) ("Sullivan"); and Bohme et al., Exp. Cell Res., 216:191-198 (1995) ("Bohme")), and vertebra (Lian et al., J. Cellular Biochem., 52:206-219 (1993) ("Lian")), limb bud mesenchymal cells in micromass cultures (Roark et al., Develop. Dynam., 200:103-116 (1994) ("Roark") and Downie et al., Dev. Biol., 162:195 (1994) ("Downie")), growth plate chondrocytes in monolayer (Rosselot et al., J. Bone Miner. Res., 9:431-439 (1994) ("Rosselot"); Gelb et al., Endocrinology, 127:1941-1947 (1990) ("Gelb"); and Crabb et al. J. Bone Mineral Res., 5:1105-1112 (1990) ("Crab")), or pellet cultures (Kato et al., Proc. Nat. Acad. Sci., 85:9552-9556 (1988) ("Kato")) have been used to characterize chondrocyte responses to exogenous factors, many of which function in an autocrine manner. From these studies has emerged a critical role for a number of growth factors, including bFGF, TGF.beta., IGF-I, and PTHrP, which are present in the growth plate and regulate chondrocyte proliferation and differentiation. The expression of these factors and their associated receptors are maturation dependent and exquisitely regulated in the growth plate (Bohme, Roark, Rosselot, Gelb, Crabb, and Hill et al., Prog. Growth Factor Res., 4:45-68 (1992)). Other studies have shown that vitamins A, C, and D are also required for chondrocyte maturation (Leboy; Sullivan; Iwamoto et al., Microscopy Res. and Technique, 28:483-491 (1994); Iwamoto et al., Exp. Cell Res., 207:413-420 (1993); Iwamoto et al., Exp. Cell Res., 205:213-224 (1993); Pacifici et al., Exp. Cell Res., 195:38-46 (1991); Shapiro et al., J. Bone Min. Res., 9:1229-1237 (1994); Corvol et al., FEBS Lett., 116:273-276 (1980); Gerstenfeld et al., Conn. Tiss. Res., 24:29-39 (1990); Schwartz et al., J. Bone Miner. Res., 4:199-207 (1989); and Suda, Calcif Tissue Int., 37:82-90 (1985)).
Transgenic mice and human cartilage defects have also provided information about endochondral ossification. Transgenic mice with deletions of the PthrP gene show premature hypertrophy of growth plate chondrocytes, demonstrating a role for PTHrP in cell proliferation and suppression of hypertrophy (Karaplis et al., Genes and Develop., 8:227-289 (1994)). Human mutations in the collagens II, IX, X, and XI are the genetic bases for mild to severe (lethal) cartilage dysplasias (Kivirikko et al., Ann. Rev. Biochem., 64:403-434 (1995)). Roles for sulfate transport (Hastabacka et al., Cell, 78:1074-1087 (1994)), sulfate metabolism (Franco et al., Cell, 81:15-25 (1995)), FGF receptor 3 (Shiang R. et al., Cell, 78:335-42 (1994)), and the transcription factor SOX9 (Wagner et al., Cell, 79:1111-1120 (1994)) in normal cartilage development have all been demonstrated by identification of genetic defects in human families.
The FGF receptor, sulfate transporters, and SOX9 are among the few examples of cellular proteins that have demonstrated roles in cartilage development. As outlined above, many of the proteins with critical roles in cartilage biology are either extracellular matrix proteins or signalling molecules. Thus, the genes and gene products instrumental to regulating the transition of chondrocytes from one stage to the next have yet to be fully characterized. Biochemical techniques used to identify matrix or intracellular components may not be sensitive enough to detect weakly or transiently expressed proteins. Furthermore, identification of cartilage defects in human or mouse mutants as a method to identify important cartilage or chondrocyte-specific proteins is limited by the number of mutants available and the labor involved in combined genetic and molecular approaches.
The present invention is directed to overcoming these and other deficiencies in the art.